The conjugation of proteins with ubiquitin involves the sequential action of proteins known as E1 (ubiquitin activating enzyme), E2s or UBCs (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). We have previously determined that RING finger proteins are in general ubiquitin ligases and have now focused on understanding issues related to the mechanism of action and substrates of specific members of this family as well as identifying inhibitors. Yeast two-hybrid screens carried out with Myriad Genetics have yielded a number of interesting interaction partners for E3 that include AO7 (RNF25), Ariadine and BRAP (IMP). For AO7 several of these interactions have been confirmed using a far-western approach, however, functional and further studies of interaction are currently ongoing. For the other E3s evaluation of interacting partners are now just underway. Our studies looking for inhibitors of Mdm2 have resulted in the identification of a family of compounds that inhibit Mdm2 E3 activity both in vitro and in cells (HLI98 family) and we are in the process of studying a related small molecule. This small molecule is water soluble and exhibits greater potency than the HLI98 compounds. This small molecule selectively kills p53-expressing cancer cells in a manner similar to the original compounds. Additionally, our screens have also resulted in the identification of an inhibitor of the ubiquitin E1 inhibitor. This inhibitor markedly deceases ubiquitin-mediated degradation of substrates as well as non-proteolytic functions of the ubiquitin system including ligand-induced activation of NfkB and ligand-mediated ubiquitination of the epidermal growth factor receptor. It also results in increased levels of p53 and in selective cell death in transformed cells expressing wild type p53. Thus, this new tool should be of great value for experimental purposes and may also serve as the basis for new therapeutics for cancer and other diseases in which perturbing functions of the ubiquitin system may be of beneficial. For information on structure-function relationships for E3 enzymes, particularly gp78, please see our other project Determining the Fate of Transmembrane Proteins.